Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2014 – vol. 118, no. 2

PHARMACY UPDATES

ECHINOCANDINS - NEW AGENTS

Cătălina Daniela Stan1, Cristina Tuchiluş2, C.I. Stan3 University of Medicine and Pharmacy “Grigore T. Popa” - Iaşi Faculty of Pharmacy 1. Drug Industry and Pharmaceutical Biotechnology Department Faculty of Medicine 2. Microbiology Department 3. Anatomy Department

ECHINOCANDINS - NEW ANTIFUNGAL AGENTS (Abstract): Over the past 10-15 years, the number of clinically available antifungal agents has increased substantially, due to rise in the number of invasive fungal infections, which are a real problem for specialists. Echi- nocandins are the new class of antifungal agents available for clinical use. This class com- prises over 20 natural echinocandins and several semisynthetic ones. Natural echinocandins are not of clinical utility due to their toxicity and low water-solubility (which does not allow obtaining parenteral pharmaceutical forms), although they have good antifungal activity against species. Consequently, semisynthetic echinocandins with minimal toxicity, good antifungal activity and high water-solubility were obtained. All echinocandins inhibit β-1,3-glucan-synthase, an essential component of the fungal cell wall. Echinocandins exhibit potent antifungal activity against key pathogenic fungi, including Candida species, Aspergil- lus species and Pneumocystis carinii. The available echinocandins lack in vitro activity against Cryptococcus neoformans. The semisynthetic echinocandins have great advantages, among which low toxicity, fast antifungal activity, favorable pharmacokinetics that allow once-daily administration. The echinocandins recently available for clinical use are: caspo- fungin, and . Keywords: MICAFUNGIN, ECHINOCANDINS, , PNEUMOCANDIN, ANIDULAFUNGIN

Antifungal agents were discovered quite antifungal agents (tab. I) (1). late, although some species of pathogenic Nowadays, the natural antifungal agents fungi were discovered in 1835 (Beauvaria most often used in therapy are: polyenic bassiana). macrolides (, , amphoter- Despite intense research carried out in icin B), spiranic antibiotics () order to discover new antifungal agents, and cyclic lipopeptide antibiotics (candins- they appeared later than the antibacterial the most popular being echinocandins). antibiotics on the pharmaceutical market. Other natural compounds are in differ- This happend because it was difficult to find ent stages of clinical testing: good antifungal agents with low toxicity. - papulacandins – glycolipid antibiotics Antifungal agents can be categorized produced by Papularia species; depending on how they are obtained as - pradimicins and benanomycins – an- synthetic antifungal agents and natural tracyclic antifungal agents isolated from

528 Echinocandins - new antifungal agents cultures of actinomycetes; gal agents isolated from cultures of acti- - nikkomycins and sardarins – antifun- nomycetes.

TABLE I Natural antifungal agents No Antifungal agent Producing miroorganism Pharmaceutical products 1. Streptomyces nodosus Fungizone, Fungilin 2. Azaserin Streptomyces spp. - 3. Candicidin Streptomyces griseus Levorin, Vanobid 4. Dermostatin Streptomyces viridogriseus Viridofulvin, Dermastatin Streptomyces cellulosae Streptomyces penticus 5. Fungicromin Streptomyces roseoluteus Cantricin Streptomyces griseus 6. Filipin Streptomyces filipenensis Filimarisin Penicillium griseofulvum Fulvicin, Grisactin, 7. Griseofulvin Penicillium janczewskii Fulcin, Grifulvin 8. Hachimycin Streptomyces hachijoensis Trichomicin, Trichonat 9. Streptomyces pimprina Primamycin 10. Lucensomycin Streptomyces lucensis Etruscomicyn, Streptomyces natalensis Pimaricin, Natacyn Pimafu- 11. Natamycin Streptomyces chattanoogensis cin, Mycophyt Streptomyces noursei, Stamicin, Nistatin, 12. Nystatin Streptomyces aureus Fungicidin, Mycostatin 13. Partricin Streptomyces aureofaciens Ayfactin Paecilomyces varioti Bainier 14. Pecilocin Supral, Variotin var. antibioticus Streptomyces coelicolor var. 15. Perimicin Aminomycin aminophilus 16. Pirrolnitrin Pseudomonas pyrrocinia Pyroace Helminthosporium siccans 17. Siccanin Tackle Drechsler 18. Trichomycin Streptomyces hachijoensis Trichomycin

Echinocandins were discovered in the patients (4, 5). Moreover, during the past 70’s and 80’s, being natural lipopeptidic decade, the incidence of invasive aspergillo- antibiotics active against the resistant strains sis has increased, especially in imunocom- of Candida and Aspergillus. These are the promised, cancer, and transplant patients (6). most recently aproved antifungal agents, Amphotericin B or are the after years of intense research (2, 3). first-line therapeutic options in the treat- Candida species are the fourth leading ment of such fungal infections, but the cause of nosocomial infections in USA, utility of amphotericin B is limited due to mortality from candidiasis is reported to be its nephrotoxicity. Fluconazole is relatively 38% in imunocompromised persons, and safe, but there are already Candida species complications could appear in over 15% of resistant to it.

529 Cătălina Daniela Stan et al.

Therefore, because invasive candidiasis cyclic hexapeptides molecules with the N- and aspergillosis are severe nosocomial linked acyl fatty acid ("side chain") with a infections, there was an urgent need for length of 14 to 18 atoms (1). new antifungal agents. The recent discov- Echinocandins are a class represented ery and development of echinocandins is an by over 20 isolated natural products, which alternative to amphotericin B and flucona- are divided into several subclasses, and zole as first-line treatment for candidiasis. many semisynthetic analogues, derived Echinocandins are large amphiphilic from natural compounds (fig. 1). R3 R4 HO O R2 NH R NH 5 N O O R1 HN OH

NH HO O R O N 6 HN R7 OH O OH

HO No Fatty acid side chain (R5) 1. Echinocandin B Linoleic acid 2. Echinocandin C Linoleic acid 3. Echinocandin D Linoleic acid 4. Aculeacin Aγ Palmitic acid 5. Mulundocandin 12-Methylmyristoic acid 6. Sporiofungin A 10,12-Dimethylmyristoic acid 7. Pneumocandin A0 10,12-Dimethylmyristoic acid 8. Pneumocandin B0 10,12-Dimethylmyristoic acid

Echinocandin R1 R2 R3 R4 R6 R7 Echinocandin B CH3 CH3 OH OH CH3 OH Echinocandin C CH3 CH3 OH OH CH3 H Echinocandin D CH3 CH3 H H CH3 H Aculeacin Aγ CH3 CH3 OH OH CH3 OH Mulundocandin H CH3 OH OH H OH Sporiofungin A CH2CONH2 CH3 OH OH H OH Pneumocandin A0 CH2CONH2 CH3 OH OH CH3 OH Pneumocandin B0 CH2CONH2 H OH OH CH3 OH Fig. 1. Natural echinocandins

All natural echinocandins are active by the fungal cell wall biosynthesis, making it inhibiting enzyme 1,3-β-D-glucan synthe- easy to lysate (1, 7). The enzyme is not tase, being selective and noncompetitive present in the cell wall of higher animals, inhibitors of the essential components of which explains the very few side effects

530 Echinocandins - new antifungal agents caused by echinocandins. due to their toxicity (they are hemolytic They have the advantage of low toxici- substances), although they have very good ty, fast antifungal activity and favorable antifungal activity against Candida strains pharmacokinetic that allows once-daily (8). They also have low water solubility, administration (having large molecules which makes them difficult for parenteral should only be given intravenously). formulations. Thus, semisynthetic com- The spectrum of natural echinocandins pounds, non-toxic, with high antifungal is limited to Candida species (spp.), but activity and enhanced water solubility were some semisynthetic analogues have a wider obtained. Today three semisynthetic echi- spectrum that includes other endogenous nocandins, caspofungin, micafungin and fungi, such as: Aspergillus spp., Pneumo- anidulafungin are licensed for clinical use cystis carinii etc. None of these compounds (9). show activity againts Cryptococcus Starting with the natural candins isolat- neoformans strains. ed from the culture medium of Glarea Echinocandin B was first isolated and lozoyensis species a semisynthetic ana- identified as the major component of the logue of pneumocandin B0, called Caspo- class (which includes echinocandin C and fungin (fig. 2) was obtained and entered the D). It was isolated from the fermentation U.S. market in 2001 by Merck (CAN- broths of various fungi of the genus Asper- CIDAS - Merck, USA, vials 50 or 70 mg gillus nidulans and Aspergillus rugulosus lyophilized powder). In Europe it was in- in 1974, at the same time by the researchers troduced in therapy in 2002 (CANCIDAS, from Ciba-Geigy, Sandoz, and Eli-Lilly. Merck & Co. Inc., USA, MK-0991). The Researchers from Toyo Jozo have isolated pharmaceutical product contains caspofun- echinocandins from aculeacin class, and gin acetate a water-soluble salt of caspo- fermentations broths of Aspergillus acule- fungin. (C52H88N10O15·2C2H4O2). atus, those from Hoechst have isolated It has a good pharmacokinetics: 100% mulundocandin from Aspergillus sydowi bioavailability, high plasma protein binding and sporiofungin class was isolated from (97%) and a half-life of 9-11 h. It has a Criptosporiopsis spp. by Sandoz research- slow hepatic metabolism and a slow elimi- ers. The pneumocandin class, with the main nation from plasma with a clearence of 10- component pneumocandin B0, was isolated 12 mL/minute. Excretion of drug is through from the fermentations broths of some hepatic (34%) and renal (41%) routes. A fungi of the genus Zalerion arboricola and small amount of caspofungin is excreted Glarea lozoyensis by Merck researchers in unchanged in urine (1.4%). 1985. Caspofungin acetate inhibits the synthe- All these compounds are substances sis of 1,3-β-D-glucan synthetase, an essen- with low water solubility, being discovered tial component of the cell wall of many quite recently. Of these, only the echi- filamentous fungi. nocandins and pneumocandins were studied It is active against , more closely for the development of sem- Candida tropicalis, Candida glabrata, Can- isynthetic derivatives. dida krusei, Candida lusitaniae, Candida Natural echinocandins and natural dubliniensis and Aspergillus species, but is pneumocandins have no therapeutic utility also active against Pneumocystis jirovecii

531 Cătălina Daniela Stan et al.

(7, 10, 11, 12). It is less active against Can- arrhizus, Fusarium spp.) and its activity dida parapsilosis and Candida guillier- against other pathogens (e.g. protozoa) is mondii, with higher minimum inhibitory undergoing clinical evaluation (13). The concentrations for these species. Caspofun- minimum inhibitory concentrations (MIC50) gin has limited activity against other fungi of caspofungin against Candida albicans (e.g. Trichosporon beigelii, Rhizopus ranges from 0.01 μg/mL to 8 μg/mL (14).

H2N

HN OH HO O O NH NH N O H2N O HN OH H3C

CH3 CH3 HO NH O O N CH3 HN HO OH O OH

HO Fig. 2. Chemical structure of Caspofungin

It is administered only intravenously 18). It is used successfully in the treatment (i.v.), the solution is reconstituted by dis- of invasive candidiasis in immuno- solving the powder exclusively in 0.9% compromised patients (19). NaCl solution. It is not compatible with Another analog of pneumocandin B0 is dextrose, so diluents containing dextrose Micafungin (fig. 3), a semisynthetic echi- have to be avoided. Caspofungin is provided nocandin obtained by conversion of the as a lyophilized powder and excipients in- natural compounds isolated from fermenta- clude sucrose, mannitol, acetic acid, and tion broths of Coleophoma empetri F-11899. sodium hydroxide. It can be stored (refriger- Micafungin is manufactured in Japan ated) for up to 24 h after reconstitution (14). and commercialized by Astellas Pharma Caspofungin (CANCIDAS) is adminis- under the trade name MYCAMINE (Mica- tered intravenously as first-line treatment fungin sodium, 50 mg or 100 mg lyophi- for Candida infections: intra-abdominal lized powder per vial). The product has abscesses, peritonitis, pleural infections received U.S. market approval from Food and in the treatment of esophageal candidi- and Drug Administration (FDA) on 16 asis. It is indicated to treat adults, adoles- March 2005. European Medicines Agency cents and children with invasive candidosis (EMA) has given approval for European and invasive aspergillosis when the patient market on 25 April 2008 (MYCAMINE, does not respond to or does not tolerate Astellas Pharma Inc., USA, FK-463). amphotericin B or . (15, 16, 17,

532 Echinocandins - new antifungal agents

OH HO O OH O OH H S N O O H2N NH OH

N O O O OH O O N

HO NH HN HO O OH HO O N H NH

O

O

Fig. 3. Chemical structure of Micafungin

Micafungin is a semisynthetic echino- ly, once daily, to treat Candida infections candin, presented as a white hygroscopic such as intra-abdominal abscesses and peri- powder. The pharmaceutical product contains tonitis (20, 21). Micafungin is approved for sodium micafungin a salt readily soluble in use in neonates, children, adults and adoles- water (C56H70N9NaO23S). cents in the treatment of invasive candi- It is active against strains of Candida dosis. It is also used, from January 2008, in albicans, Candida glabrata, Candida krusei the prophylaxis of candida infection in pa- and Candida tropicalis. It is less active tients undergoing allogeneic hematopoietic against Candida parapsilosis and Candida stem cell transplantation or patients who are guilliermondii, with higher minimum inhibi- expected to have granulocytopenia (22). In tory concentrations for these species. The addition, micafungin is used in the treatment minimum inhibitory concentrations (MIC50) of esophageal candidosis in adults. of micafungin against Candida albicans It has few side effects, like other sem- ranges from 0.01 μg/mL to 0.5 μg/mL (14). isynthetic echinocandins being very well Micafungin is characterized by linear tolerated. However, patients who develop pharmacokinetics. It is metabolized into abnormal liver function tests during treat- three metabolites which are excreted slow- ment with micafungin should be evaluated ly over many days, mainly in the bile. It is for the risk/benefit ratio. highly bound to plasma protein (99%), and The vials contain sodium micafungin the half-life is 10-17 hours. Micafugin is and excipients which include lactose, citric eliminated via digestive (40%) and renal acid and sodium hydroxide. The lyophi- (15%) routes. lized powder is ready for reconstitution by Micafungin is administered intravenous- dissolving with NaCl 0.9% or dextrose 5%.

533 Cătălina Daniela Stan et al.

Reconstituted solution is stable at room An analogue of echinocandin B is temperatures for 48 h, if protected from Anidulafungin (fig. 4), marketed by Pfizer light. (U.K.).

H HO O H H C OH N C C H NH O O C H HO C HO C NH HO O NH C O C C C H H H H OH C H C OH O NH NH C C HO C N H O C H H O H OH H

Fig. 4. Chemical structure of Anidulafungin

Pfizer received U.S. market approval bicans, Candida glabrata, Candida krusei from FDA for anidulafungin under the and Candida tropicalis. It is less active trade name ERAXIS (50 mg or 100 mg against Candida parapsilosis and Candida vials containing lyophilized powder) in guilliermondii, with higher minimum inhibi- February 2006. EMA has given approval tory concentrations for these species (23). for European market on November 2007 The minimum inhibitory concentrations for anidulafungin under the trade name (MIC50) of anidulafungin against Candida ECALTA (50 mg or 100 mg vial containing albicans ranges from 0.01 μg/mL to 8 μg/mL lyophilized powder, Pfizer Inc. UK). (14). On other fungi anidulagfungin activity It is a semisynthetic echinocandin ob- is limited (e.g. Acremonium spp., Phialopho- tained by converting natural echinocandin ra spp., Rhizopus spp., Fusarium spp.). B isolated from Aspergillus nidulans spe- Anidulafungin is slowly metabolized in- cies. It was initially researched by Eli Lilly, to an inactive open-ring peptide which is clinically developed by Vicuron company, excreted in the bile. No liver metabolism and later marketed by Pfizer. has been observed. It is highly bound to It is a white powder (C58H73N7O17), plasma protein (99%), and the half-life is freely soluble in water, with an alkoxy- 0.5-1 hour. It is eliminated via digestive triphenyl side chain which intercalates with (30%) and renal (1%) routes. the phosphor-lipid bilayer of the cell mem- Anidulafungin is administered intrave- brane. nously to treat invasive candidosis, inva- It is active against strains of Candida al- sive aspergillosis in adults who are not

534 Echinocandins - new antifungal agents granulocytopenic, but not in children or favorable pharmacokinetics, which allow adolescents below the age of 18 years (23). once-daily intravenous administration. It is administered once-daily, the duration They exhibit potent antifungal activity of treatment depending on patient response, against key pathogenic fungi, including usually at least 14 days. It has few side Candida species, Aspergillus species and effects, like other semisynthetic echino- Pneumocystis species. All clinical trials candins being very well tolerated. It can be demonstrate their efficacy in the treatment used in patients with renal and hepatic of invasive candidosis and invasive asper- impairment (14, 23, 24). gillosis in patients not responding to other The vials contain anidulafungin and ex- antifungal agents. They can be used suc- cipients as a lyophilized powder. The excip- cessfully in the treatment of invasive can- ients include fructose, tartric acid, manitol, didiasis in immunocompromised patients, sodium hydroxide and hydrochloric acid. or in patients with renal and hepatic im- The lyophilized powder is ready for recon- pairment. stitution with water for injections and sub- The echinocandins recently available sequently diluted with NaCl 0.9% or glucose for clinical use are: caspofungin, mica- 5% for infusion. Reconstituted solution is fungin and anidulafungin. stable at room temperatures for 24 h. Their safe use, route of administration, Echinocandins are the newest antifungal fewest interactions with other drugs and agents available for clinical use. The sem- their spectrum of action make echino- isynthetic echinocandins have great ad- candins a class of antifungal agents with a vantages such as, low toxicity, great anti- secure future, although today they are less fungal activity, high water solubility, and accessible due to the cost.

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